Biosynthesis of the assimilatory NADPH-nitrate reductase in Aspergillus nidulans is directed by the structural gene (niaD ion) and five cofactor loci (cnx ABC, E, F, G and H). The precise role of the cnx loci biosynthesis of the enzyme is unknown. This project will make use of the wild type and selected mutant strains to help discern the extent to which the niaD and the cnx directed promoters are involved in the form and function of the native multimer. Direct evidence for de novo synthesis of the nitrate reductase and especially the niaD directed promoter will be obtained by use of isotopically tagged amino acids such as (3h)tyrosine, (14C)tyrosine, (3H)lysine, (14C)lysine, and (35S)cysteine as well as Na2 35SO4. Enzyme will be isolated from samples of wild type and mutant mycelium (removed at appropriate intervals during growth) and its quantity of label will be measured. Direct evidence for the structural involvement of the cnxH locus will be obtained by the use of a dual label procedure and heterocaryon complementation. Two mutant strains, one contributing the structural gene and the other contributing a normal cnxH directed protein, will be grown separately in the presense of (3H)tyr or (14C)tyr respectively. The labelled mutant mycelia will be washed free of label and transferred to a medium where nitrate reductase must be synthesized via genetic complementation. Hybrid enzyme will be obtained since neither mutant can synthesize a functional nitrate reductase alone. The ratio of 14C/3H in the enzyme should reflect the structural contribution of the cnxH mutant partner. The use of a temperature sensitive cnxH mutant is planned to confirm the association of the labelled promoter and thermolabile catalytic function. The proposed research should reveal more information on the hierarchy of genes functioning during assembly of heteromultimeric proteins in the eucaryotic cell.